DNA stretching induces Cas9 off-target activity.
Identifieur interne : 000B61 ( Main/Exploration ); précédent : 000B60; suivant : 000B62DNA stretching induces Cas9 off-target activity.
Auteurs : Matthew D. Newton [Royaume-Uni] ; Benjamin J. Taylor [Royaume-Uni] ; Rosalie P C. Driessen [Pays-Bas] ; Leonie Roos [Royaume-Uni] ; Nevena Cvetesic [Royaume-Uni] ; Shenaz Allyjaun [Royaume-Uni] ; Boris Lenhard [Royaume-Uni] ; Maria Emanuela Cuomo [Royaume-Uni] ; David S. Rueda [Royaume-Uni]Source :
- Nature structural & molecular biology [ 1545-9985 ] ; 2019.
Descripteurs français
- KwdFr :
- ADN viral (génétique), ADN viral (métabolisme), ARN guide (génétique), Bactériophage lambda (génétique), Clivage de l'ADN, Clustered regularly interspaced short palindromic repeats (génétique), Escherichia coli (virologie), Microfluidique, Microscopie confocale, Pinces optiques, Streptococcus pyogenes (enzymologie), Systèmes CRISPR-Cas (génétique), Transfert d'énergie par résonance de fluorescence, Édition de gène.
- MESH :
- enzymologie : Streptococcus pyogenes.
- génétique : ADN viral, ARN guide, Bactériophage lambda, Clustered regularly interspaced short palindromic repeats, Systèmes CRISPR-Cas.
- métabolisme : ADN viral.
- virologie : Escherichia coli.
- Clivage de l'ADN, Microfluidique, Microscopie confocale, Pinces optiques, Transfert d'énergie par résonance de fluorescence, Édition de gène.
English descriptors
- KwdEn :
- Bacteriophage lambda (genetics), CRISPR-Associated Protein 9 (metabolism), CRISPR-Cas Systems (genetics), Clustered Regularly Interspaced Short Palindromic Repeats (genetics), DNA Cleavage, DNA, Viral (genetics), DNA, Viral (metabolism), Escherichia coli (virology), Fluorescence Resonance Energy Transfer, Gene Editing, Microfluidics, Microscopy, Confocal, Optical Tweezers, RNA, Guide (genetics), Streptococcus pyogenes (enzymology).
- MESH :
- chemical , genetics : DNA, Viral, RNA, Guide.
- chemical , metabolism : CRISPR-Associated Protein 9, DNA, Viral.
- enzymology : Streptococcus pyogenes.
- genetics : Bacteriophage lambda, CRISPR-Cas Systems, Clustered Regularly Interspaced Short Palindromic Repeats.
- virology : Escherichia coli.
- DNA Cleavage, Fluorescence Resonance Energy Transfer, Gene Editing, Microfluidics, Microscopy, Confocal, Optical Tweezers.
Abstract
CRISPR/Cas9 is a powerful genome-editing tool, but spurious off-target edits present a barrier to therapeutic applications. To understand how CRISPR/Cas9 discriminates between on-targets and off-targets, we have developed a single-molecule assay combining optical tweezers with fluorescence to monitor binding to λ-DNA. At low forces, the Streptococcus pyogenes Cas9 complex binds and cleaves DNA specifically. At higher forces, numerous off-target binding events appear repeatedly at the same off-target sites in a guide-RNA-sequence-dependent manner, driven by the mechanical distortion of the DNA. Using single-molecule Förster resonance energy transfer (smFRET) and cleavage assays, we show that DNA bubbles induce off-target binding and cleavage at these sites, even with ten mismatches, as well as at previously identified in vivo off-targets. We propose that duplex DNA destabilization during cellular processes (for example, transcription, replication, etc.) can expose these cryptic off-target sites to Cas9 activity, highlighting the need for improved off-target prediction algorithms.
DOI: 10.1038/s41594-019-0188-z
PubMed: 30804513
Affiliations:
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Le document en format XML
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Newton</name><affiliation wicri:level="3"><nlm:affiliation>Molecular Virology, Department of Medicine, Imperial College London, London, UK.</nlm:affiliation><country xml:lang="fr">Royaume-Uni</country><wicri:regionArea>Molecular Virology, Department of Medicine, Imperial College London, London</wicri:regionArea><placeName><settlement type="city">Londres</settlement><region type="country">Angleterre</region><region type="région" nuts="1">Grand Londres</region></placeName></affiliation></author><author><name sortKey="Taylor, Benjamin J" sort="Taylor, Benjamin J" uniqKey="Taylor B" first="Benjamin J" last="Taylor">Benjamin J. Taylor</name><affiliation wicri:level="1"><nlm:affiliation>Discovery Sciences, IMED Biotech Unit, Astra Zeneca, Cambridge, UK.</nlm:affiliation><country xml:lang="fr">Royaume-Uni</country><wicri:regionArea>Discovery Sciences, IMED Biotech Unit, Astra Zeneca, Cambridge</wicri:regionArea><wicri:noRegion>Cambridge</wicri:noRegion></affiliation></author><author><name sortKey="Driessen, Rosalie P C" sort="Driessen, Rosalie P C" uniqKey="Driessen R" first="Rosalie P C" last="Driessen">Rosalie P C. Driessen</name><affiliation wicri:level="3"><nlm:affiliation>LUMICKS BV, Amsterdam, The Netherlands.</nlm:affiliation><country xml:lang="fr">Pays-Bas</country><wicri:regionArea>LUMICKS BV, Amsterdam</wicri:regionArea><placeName><settlement type="city">Amsterdam</settlement><region nuts="2" type="province">Hollande-Septentrionale</region></placeName></affiliation></author><author><name sortKey="Roos, Leonie" sort="Roos, Leonie" uniqKey="Roos L" first="Leonie" last="Roos">Leonie Roos</name><affiliation wicri:level="3"><nlm:affiliation>Computational Regulatory Genomics, MRC-London Institute of Medical Sciences, London, UK.</nlm:affiliation><country xml:lang="fr">Royaume-Uni</country><wicri:regionArea>Computational Regulatory Genomics, MRC-London Institute of Medical Sciences, London</wicri:regionArea><placeName><settlement type="city">Londres</settlement><region type="country">Angleterre</region><region type="région" nuts="1">Grand Londres</region></placeName></affiliation></author><author><name sortKey="Cvetesic, Nevena" sort="Cvetesic, Nevena" uniqKey="Cvetesic N" first="Nevena" last="Cvetesic">Nevena Cvetesic</name><affiliation wicri:level="3"><nlm:affiliation>Computational Regulatory Genomics, MRC-London Institute of Medical Sciences, London, UK.</nlm:affiliation><country xml:lang="fr">Royaume-Uni</country><wicri:regionArea>Computational Regulatory Genomics, MRC-London Institute of Medical Sciences, London</wicri:regionArea><placeName><settlement type="city">Londres</settlement><region type="country">Angleterre</region><region type="région" nuts="1">Grand Londres</region></placeName></affiliation></author><author><name sortKey="Allyjaun, Shenaz" sort="Allyjaun, Shenaz" uniqKey="Allyjaun S" first="Shenaz" last="Allyjaun">Shenaz Allyjaun</name><affiliation wicri:level="3"><nlm:affiliation>Molecular Virology, Department of Medicine, Imperial College London, London, UK.</nlm:affiliation><country xml:lang="fr">Royaume-Uni</country><wicri:regionArea>Molecular Virology, Department of Medicine, Imperial College London, London</wicri:regionArea><placeName><settlement type="city">Londres</settlement><region type="country">Angleterre</region><region type="région" nuts="1">Grand Londres</region></placeName></affiliation></author><author><name sortKey="Lenhard, Boris" sort="Lenhard, Boris" uniqKey="Lenhard B" first="Boris" last="Lenhard">Boris Lenhard</name><affiliation wicri:level="3"><nlm:affiliation>Computational Regulatory Genomics, MRC-London Institute of Medical Sciences, London, UK.</nlm:affiliation><country xml:lang="fr">Royaume-Uni</country><wicri:regionArea>Computational Regulatory Genomics, MRC-London Institute of Medical Sciences, London</wicri:regionArea><placeName><settlement type="city">Londres</settlement><region type="country">Angleterre</region><region type="région" nuts="1">Grand Londres</region></placeName></affiliation></author><author><name sortKey="Cuomo, Maria Emanuela" sort="Cuomo, Maria Emanuela" uniqKey="Cuomo M" first="Maria Emanuela" last="Cuomo">Maria Emanuela Cuomo</name><affiliation wicri:level="1"><nlm:affiliation>Discovery Sciences, IMED Biotech Unit, Astra Zeneca, Cambridge, UK. emanuela.cuomo@astrazeneca.com.</nlm:affiliation><country xml:lang="fr">Royaume-Uni</country><wicri:regionArea>Discovery Sciences, IMED Biotech Unit, Astra Zeneca, Cambridge</wicri:regionArea><wicri:noRegion>Cambridge</wicri:noRegion></affiliation></author><author><name sortKey="Rueda, David S" sort="Rueda, David S" uniqKey="Rueda D" first="David S" last="Rueda">David S. Rueda</name><affiliation wicri:level="3"><nlm:affiliation>Molecular Virology, Department of Medicine, Imperial College London, London, UK. david.rueda@imperial.ac.uk.</nlm:affiliation><country xml:lang="fr">Royaume-Uni</country><wicri:regionArea>Molecular Virology, Department of Medicine, Imperial College London, London</wicri:regionArea><placeName><settlement type="city">Londres</settlement><region type="country">Angleterre</region><region type="région" nuts="1">Grand Londres</region></placeName></affiliation></author></analytic><series><title level="j">Nature structural & molecular biology</title><idno type="eISSN">1545-9985</idno><imprint><date when="2019" type="published">2019</date></imprint></series></biblStruct></sourceDesc></fileDesc><profileDesc><textClass><keywords scheme="KwdEn" xml:lang="en"><term>Bacteriophage lambda (genetics)</term><term>CRISPR-Associated Protein 9 (metabolism)</term><term>CRISPR-Cas Systems (genetics)</term><term>Clustered Regularly Interspaced Short Palindromic Repeats (genetics)</term><term>DNA Cleavage</term><term>DNA, Viral (genetics)</term><term>DNA, Viral (metabolism)</term><term>Escherichia coli (virology)</term><term>Fluorescence Resonance Energy Transfer</term><term>Gene Editing</term><term>Microfluidics</term><term>Microscopy, Confocal</term><term>Optical Tweezers</term><term>RNA, Guide (genetics)</term><term>Streptococcus pyogenes (enzymology)</term></keywords><keywords scheme="KwdFr" xml:lang="fr"><term>ADN viral (génétique)</term><term>ADN viral (métabolisme)</term><term>ARN guide (génétique)</term><term>Bactériophage lambda (génétique)</term><term>Clivage de l'ADN</term><term>Clustered regularly interspaced short palindromic repeats (génétique)</term><term>Escherichia coli (virologie)</term><term>Microfluidique</term><term>Microscopie confocale</term><term>Pinces optiques</term><term>Streptococcus pyogenes (enzymologie)</term><term>Systèmes CRISPR-Cas (génétique)</term><term>Transfert d'énergie par résonance de fluorescence</term><term>Édition de gène</term></keywords><keywords scheme="MESH" type="chemical" qualifier="genetics" xml:lang="en"><term>DNA, Viral</term><term>RNA, Guide</term></keywords><keywords scheme="MESH" type="chemical" qualifier="metabolism" xml:lang="en"><term>CRISPR-Associated Protein 9</term><term>DNA, Viral</term></keywords><keywords scheme="MESH" qualifier="enzymologie" xml:lang="fr"><term>Streptococcus pyogenes</term></keywords><keywords scheme="MESH" qualifier="enzymology" xml:lang="en"><term>Streptococcus pyogenes</term></keywords><keywords scheme="MESH" qualifier="genetics" xml:lang="en"><term>Bacteriophage lambda</term><term>CRISPR-Cas Systems</term><term>Clustered Regularly Interspaced Short Palindromic Repeats</term></keywords><keywords scheme="MESH" qualifier="génétique" xml:lang="fr"><term>ADN viral</term><term>ARN guide</term><term>Bactériophage lambda</term><term>Clustered regularly interspaced short palindromic repeats</term><term>Systèmes CRISPR-Cas</term></keywords><keywords scheme="MESH" qualifier="métabolisme" xml:lang="fr"><term>ADN viral</term></keywords><keywords scheme="MESH" qualifier="virologie" xml:lang="fr"><term>Escherichia coli</term></keywords><keywords scheme="MESH" qualifier="virology" xml:lang="en"><term>Escherichia coli</term></keywords><keywords scheme="MESH" xml:lang="en"><term>DNA Cleavage</term><term>Fluorescence Resonance Energy Transfer</term><term>Gene Editing</term><term>Microfluidics</term><term>Microscopy, Confocal</term><term>Optical Tweezers</term></keywords><keywords scheme="MESH" xml:lang="fr"><term>Clivage de l'ADN</term><term>Microfluidique</term><term>Microscopie confocale</term><term>Pinces optiques</term><term>Transfert d'énergie par résonance de fluorescence</term><term>Édition de gène</term></keywords></textClass></profileDesc></teiHeader><front><div type="abstract" xml:lang="en">CRISPR/Cas9 is a powerful genome-editing tool, but spurious off-target edits present a barrier to therapeutic applications. To understand how CRISPR/Cas9 discriminates between on-targets and off-targets, we have developed a single-molecule assay combining optical tweezers with fluorescence to monitor binding to λ-DNA. At low forces, the Streptococcus pyogenes Cas9 complex binds and cleaves DNA specifically. At higher forces, numerous off-target binding events appear repeatedly at the same off-target sites in a guide-RNA-sequence-dependent manner, driven by the mechanical distortion of the DNA. Using single-molecule Förster resonance energy transfer (smFRET) and cleavage assays, we show that DNA bubbles induce off-target binding and cleavage at these sites, even with ten mismatches, as well as at previously identified in vivo off-targets. We propose that duplex DNA destabilization during cellular processes (for example, transcription, replication, etc.) can expose these cryptic off-target sites to Cas9 activity, highlighting the need for improved off-target prediction algorithms.</div></front></TEI><affiliations><list><country><li>Pays-Bas</li><li>Royaume-Uni</li></country><region><li>Angleterre</li><li>Grand Londres</li><li>Hollande-Septentrionale</li></region><settlement><li>Amsterdam</li><li>Londres</li></settlement></list><tree><country name="Royaume-Uni"><region name="Angleterre"><name sortKey="Newton, Matthew D" sort="Newton, Matthew D" uniqKey="Newton M" first="Matthew D" last="Newton">Matthew D. Newton</name></region><name sortKey="Allyjaun, Shenaz" sort="Allyjaun, Shenaz" uniqKey="Allyjaun S" first="Shenaz" last="Allyjaun">Shenaz Allyjaun</name><name sortKey="Cuomo, Maria Emanuela" sort="Cuomo, Maria Emanuela" uniqKey="Cuomo M" first="Maria Emanuela" last="Cuomo">Maria Emanuela Cuomo</name><name sortKey="Cvetesic, Nevena" sort="Cvetesic, Nevena" uniqKey="Cvetesic N" first="Nevena" last="Cvetesic">Nevena Cvetesic</name><name sortKey="Lenhard, Boris" sort="Lenhard, Boris" uniqKey="Lenhard B" first="Boris" last="Lenhard">Boris Lenhard</name><name sortKey="Roos, Leonie" sort="Roos, Leonie" uniqKey="Roos L" first="Leonie" last="Roos">Leonie Roos</name><name sortKey="Rueda, David S" sort="Rueda, David S" uniqKey="Rueda D" first="David S" last="Rueda">David S. Rueda</name><name sortKey="Taylor, Benjamin J" sort="Taylor, Benjamin J" uniqKey="Taylor B" first="Benjamin J" last="Taylor">Benjamin J. Taylor</name></country><country name="Pays-Bas"><region name="Hollande-Septentrionale"><name sortKey="Driessen, Rosalie P C" sort="Driessen, Rosalie P C" uniqKey="Driessen R" first="Rosalie P C" last="Driessen">Rosalie P C. Driessen</name></region></country></tree></affiliations></record>Pour manipuler ce document sous Unix (Dilib)
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